Abstract
The in vitro differentiation of human embryonic stem cells (hESCs) offers a model system to explore human development. Humans with mutations in the transcription factor Aristaless Related Homeobox (ARX) often suffer from the syndrome X-linked lissencephaly with ambiguous genitalia (XLAG), affecting many cell types including those of the pancreas. Indeed, XLAG pancreatic islets lack glucagon and pancreatic polypeptide-positive cells but retain somatostatin, insulin, and ghrelin-positive cells. To further examine the role of ARX in human pancreatic endocrine development, we utilized genomic editing in hESCs to generate deletions in ARX. ARX knockout hESCs retained pancreatic differentiation capacity and ARX knockout endocrine cells were biased toward somatostatin-positive cells (94% of endocrine cells) with reduced pancreatic polypeptide (rarely detected), glucagon (90% reduced) and insulin-positive (65% reduced) lineages. ARX knockout somatostatin-positive cells shared expression patterns with human fetal and adult δ-cells. Differentiated ARX knockout cells upregulated PAX4, NKX2.2, ISL1, HHEX, PCSK1, PCSK2 expression while downregulating PAX6 and IRX2. Re-expression of ARX in ARX knockout pancreatic progenitors reduced HHEX and increased PAX6 and insulin expression following differentiation. Taken together these data suggest that ARX plays a key role in pancreatic endocrine fate specification of pancreatic polypeptide, somatostatin, glucagon and insulin positive cells from hESCs.
Highlights
The most effective therapy for type 1 diabetes is cadaveric human islet transplantation, the demand for donor tissue outpaces the availability of cells
To examine the role of Aristaless Related Homeobox (ARX) in pancreatic endocrine differentiation, we generated two independent ARX knockout (ARX ko) human embryonic stem cells (hESCs) clones with genomic deletions in exon 1, such that all protein components from exons 2–5 are lost (Fig 1A)
This was accomplished by targeted genomic editing using a zinc-finger nuclease pair that stimulated double stranded DNA breaks in the single copy of ARX in male (XY) CA1S hESCs
Summary
The most effective therapy for type 1 diabetes is cadaveric human islet transplantation, the demand for donor tissue outpaces the availability of cells. Based on this need, the use of pluripotent stem cells and their differentiated progeny is being explored as a potential source of insulin secreting β-cells for transplantation [1]. Many in vitro differentiation protocols generate polyhormonal endocrine cells that co-express insulin, glucagon and the transcription factor Aristaless Related Homeobox (ARX) [2,3,4,5,6,7]. ARX in hESCs and Human Pancreatic Development PLOS ONE | DOI:10.1371/journal.pone.0144100 December 3, 2015
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